Ribotoxins are potent inhibitors of protein synthesis. They are believed to act as enzymes on the 28S rRNA of the eucaryotic ribosome and can be divided into two classes (Jiminez, A et al., 1985, Ann. Rev Microbiol., 39, 649-672, and Wool, I. G. et al., 1990, in Hill, W. E. et al. (ed.) The Ribosome Structure, Function & Evolution, American Society of Microbiology, Washington D.C., 203-214). Restrictocin, mitogillin and .alpha.-sarcin belong to the class which cleaves a single phosphodiester bond. Ricin belongs to the other class which cleaves an N-glycosidic linkage between base and ribose. The nucleotide sequence of the restrictocin gene has been described by Lamy, B. & Davies, J. in Nucleic Acids Research, 1991, 19, 1001-1006.
Immunotoxins are an example of a conjugate formed between two partners namely a toxin and an immunoglobulin or antibody. The partners are usually joined via a chemical linker but may be joined directly. Ribotoxins such as for example restrictocin may be used as the toxin component of immunotoxins, linked to the antibody via a variety of chemical linkers. Known methods involve derivatisation of the restrictocin with chemicals such as iminothiolane (although other derivatising agents, eg. N-succinimidyl-3-(2-pyridyldithio)propionate (hereinafter abbreviated SPDP) may be used) to introduce a sulphydryl functionality and to form a disulphide cross-linked structure with an appropriately derivatised antibody.
Due to the inherent random nature of the chemical derivatisation at, primarily (although not necessarily solely), amino groups such as for example on the restrictocin molecule of which there are 17 including the alpha amino group, a heterogeneous product results comprising underivatised restrictocin, mono, di, tri etc derivatised molecules which may result in a complex product profile following conjugation to antibody (1,2,3 etc moles of restrictocin/mole antibody or, potentially, 1,2,3 etc moles of antibody/mole Restrictocin). Such a heterogeneous product undesirably produces batch to batch variability and reduced overall yield. Batch to batch variability is particularly undesirable in the pharmaceutical field where the product may be for example an immunotoxin wherein the antibody is selective for tumours and is used in cancer therapy.
In addition chemical derivatisation may derivatise residues close to the active site of the molecule so compromising biological activity of the derivatised toxin.